The present invention relates to the discovery, identification, and characterization of novel human polynucleotides that encode proteins that share sequence similarity with lipoxygenases. The invention encompasses the described polynucleotides, host cell expression systems, the encoded proteins, fusion proteins, polypeptides and peptides, antibodies to the encoded proteins and peptides, and genetically engineered animals that lack the disclosed genes or over-express the disclosed genes, or antagonists and agonists of the proteins, and other compounds that modulate the expression or activity of the proteins encoded by the disclosed polynucleotides that can be used for diagnosis, drug screening, clinical trial monitoring, or the treatment of physiological or behavioral disorders.
Lipoxygenases are enzymes that mediate the oxidation of lipid substrates. As such, lipoxygenases are involved in the synthesis of leukotrienes. Leukotrienes influence a variety of biological processes, and can serve as, inter alia, potent chemotactic agents, and mediators of inflammation, smooth muscle contraction, etc. Accordingly, lipoxygenases represent a key target for the regulation of a variety of biological pathways and conditions.
The present invention relates to the discovery, identification, and characterization of nucleotides that encode novel human lipoxygenase proteins, and the corresponding amino acid sequences of these proteins. The novel human proteins (NHPs) described for the first time herein share structural similarity with animal and plant lipoxygenase proteins. As such, the novel genes represent a new class of lipoxygenase proteins with a range of homologues and orthologs that transcend a broad range of phyla and species.
The invention comprises (a) polypeptides with SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28; (b) homologues and allelic variants of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28; (c) fragments of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28 of any size, for example, from 4 amino acids to less than the full-length of a polypeptide of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, or 28 and any number between; (d) fragments of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28 that correspond to a functional domain (for example, a catalytic domain, a signal sequence, a ligand binding domain, a regulatory domain, etc.); (e) fusion proteins comprising a polypeptide sequence of any one of (a) through (d); (f) mutant polypeptides (including engineered and naturally occurring mutants) comprising a polypeptide sequence of any one of (a) through (d), including, but not limited to, deletion mutants, insertion mutants, substitution mutants, and mutant polypeptides in which all or a part of at least one of the domains is deleted or altered (e.g., a mutant of the active site with altered substrate specificity).
The invention further comprises (g) polynucleotides with SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29; (h) polynucleotides encoding any one of the polypeptides of the invention including, but not limited to, polypeptides specifically described in (a) through (f) above; (i) polynucleotides capable of hybridizing to a second polynucleotide that is complementary to a polynucleotide described in (g) and/or (h) above under conditions of low, medium, or high stringency; (j) oligonucleotides corresponding to a segment of a polynucleotide described in (g) through (i) above and such oligonucleotides having any size from 2 nucleotides through less than the full-length polynucleotide and any length inbetween.
In certain embodiments, the novel human nucleic acid sequences described herein, encode proteins/open reading frames (ORFs) of 711, 489, 556, 334, 615, 460, 291, 69, 139, 195, 110, 867, 645, and 771 amino acids in length (see SEQ ID NOS:2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, and 28 respectively).
The invention further comprises antibodies to any one of the polypeptides or polynucleotides of the invention. The invention also comprises host cells that are engineered to contain and/or express any one of the polynucleotides and/or polypeptides of the invention.
The invention also comprises agonists and antagonists of the described NHPs, including small molecules, large molecules, mutant NHPs, or portions thereof that compete with native NHP, and antibodies. The invention further comprises nucleotide sequences that can be used to inhibit the expression of the described NHPs (e.g., antisense and ribozyme oligonucleotides and/or polynucleotides, and gene or regulatory sequence replacement constructs) or to enhance the expression of the described NHP genes (e.g., expression constructs that place the described gene under the control of a strong promoter system), and transgenic animals that express a NHP transgene, or xe2x80x9cknock-outsxe2x80x9d (which can be conditional) that do not express functional NHP.
Further, the present invention also relates to methods for identifying compounds that modulate, i.e., act as agonists or antagonists of, NHP expression and/or NHP product activity that utilize purified preparations of the described NHPs and/or NHP product, or cells expressing the same. Such compounds can be used as therapeutic agents for the treatment of any of a wide variety of symptoms associated with biological disorders or imbalances.
The Sequence Listing provides the sequences of 14 lipoxygenase-like ORFs that are encoded by the described NHP polynucleotides (SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29) and the amino acid sequences (SEQ ID NOS:2, 4, 6, 5 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28) encoded thereby.
Lipoxygenases oxidize, or oxygenate, lipids to produce leukotrienes. Depending on the leukotriene synthesized, a wide variety of biological functions can be affected. Typically, leukotrienes will bind cognate receptors an trigger a biological effect (such as, for example, signal transduction). Interfering with lipoxygenase activity ultimately effects leukotriene production and downstream leukotriene-mediated processes. Alternatively, enhancing lipoxygenase activity in vivo, can boost the effects/activity levels the corresponding biological processes. Various lipoxygenase activities can be found in a variety of cells and tissues in both animals and plants. Three predominant types of lipoxygenases include the 5-, 12-, and 15-lipoxygenases, and each type of lipoxygenase can have additional forms depending upon the tissues or cells in which they are expressed.
The 5-, 12-, and 15-lipoxygenases, and the leukotrienes they produce, have been implicated with a variety of diseases and disorders. Given that leukotrienes can modulate inflammatory reactions, they have been associated with a spectrum of mammalian diseases including, but not limited to, asthma, eye diseases, anaphylaxis, lung disease, hematological disorders, infectious diseases, granulomatosis, abscess, pacreatitis, prostatitis, hepatitis, atherosclerosis, heart disease, graft rejection, thrombosis, restenosis, ulcers, kidney disease, hypertension, dermatoses, cramping, autoimmune disorders (lupus, scleroderma, Crohn""s disease, rheumatoid arthritis, etc.), granulomatosis, hyperproliferative diseases, cancer, nausea, headache, metastases, inflammatory bowel disorder, allergy, cancer, arthritis, eczema, melanoma, erythema, acne, psoriasis, shingles, infectious disease, and diabetes. Accordingly, one embodiment of the present invention are processes for identifying compounds useful for the treatment of one or more of the above diseases and disorders that include the use of one or more of the described lipoxygenase-like genes, proteins, or a novel portion thereof.
Given the biological importance of lipoxygenases, the genes encoding such proteins (and the proteins encoded thereby as well as inhibitors thereof) have been subjected to intense scientific/commercial scrutiny (see, for example, U.S. Pat. Nos. 5,036,105, 5,162,365, 5,504,097, 5,066,679, 5,830,453, 4,761,403, 5,589,506, 5,026,729, and 5,861,268) (all of which are herein incorporated by reference in their entirety).
The presently described NHPs share significant similarity with previously described human lipoxygenases. Expression studies using RT-PCR detect NHP transcripts in, inter alia, neural tissue (i.e., brain, spinal cord, etc.), skin, testis, prostate, adrenal gland, cervix, salivary gland, pancreas, heart, lymphoid cells (lymph node, spleen, thymus), and mammary glands. Northern analysis showed a predominant signal in testis, with less predominant, but longer, transcripts detectable in testis, lymph node, and spinal cord. A full length cDNA of a NHP coding region (with 5xe2x80x2 and 3xe2x80x2 extensions) was isolated from a human brain cDNA library (Edge BioSystems, Gaithersburg, Md.) and sequenced (SEQ ID NO: 29). A possible murine ortholog of the described NHPs is predominantly expressed in skin (Kinzig et al., 1999, Genomics 58:158-164).
The invention encompasses the use of the described NHP nucleotides, NHPs and peptides, as well as antibodies, preferably monoclonal antibodies, or binding fragments, domains, or fusion proteins thereof, or anti-idiotypic variants derived therefrom, that bind NHPs, other antagonists that inhibit binding activity or expression, or agonists that activate NHP activity or increase NHP expression, in the diagnosis and/or treatment of disease.
In particular, the invention described in the subsections below encompasses NHP polypeptides or peptides corresponding to functional domains of NHPs, mutated, truncated or deleted NHPs (e.g., NHPs missing one or more functional domains or portions thereof), NHP fusion proteins (e.g., a NHP or a functional domain of a NHP fused to an unrelated protein or peptide such as an immunoglobulin constant region, i.e., IgFc), nucleotide sequences encoding such products, and host cell expression systems that can produce such NHP products.
The invention also encompasses antibodies and anti-idiotypic antibodies (including Fab fragments), antagonists and agonists of the NHP, as well as compounds or nucleotide constructs that inhibit expression of a NHP gene (transcription factor inhibitors, antisense and ribozyme molecules, or gene or regulatory sequence replacement constructs), or promote expression of NHP (e.g., expression constructs in which NHP coding sequences are operatively associated with expression control elements such as promoters, promoter/enhancers, etc.). The invention also relates to as host cells and animals genetically engineered to express the NHPs (or mutant variants thereof) or to inhibit or xe2x80x9cknock-outxe2x80x9d expression of an animal homolog of an endogenous NHP gene.
The NHPs or peptides, NHP fusion proteins, NHP nucleotide sequences, antibodies, antagonists and agonists can be useful for the detection of mutant NHPs or inappropriately expressed NHPs for the diagnosis of disease. The NHP proteins or peptides, NHP fusion proteins, NHP nucleotide sequences, host cell expression systems, antibodies, antagonists, agonists and genetically engineered cells and animals can be used for screening for drugs (or high throughput screening of combinatorial libraries) effective in the treatment of the symptomatic or phenotypic manifestations of perturbing the normal function of NHP in the body.
The use of engineered host cells and/or animals offers an advantage in that such systems allow for both the identification of compounds that interact with an NHP, and also provide information regarding the biological significance of the NHP.
Finally, NHP products (especially soluble derivatives such as peptides corresponding to the NHP), and NHP fusion protein products (such as NHP-Ig fusion proteins, i.e., fusions of a NHP, or a domain of a NHP, to an IgFc), antibodies and anti-idiotypic antibodies (including Fab fragments), antagonists or agonists (including compounds that modulate signal transduction which may act on downstream targets in a NHP-associated leukotriene pathway) can be used to directly treat diseases or disorders.
Nucleotide constructs encoding such NHP products can be delivered to host cells that subsequently express the products in vivo; these genetically engineered cells function as xe2x80x9cbioreactorsxe2x80x9d in the body delivering a continuous supply of a NHP, a NHP peptide, or a NHP fusion protein to the body. Nucleotide constructs encoding functional NHPs, mutant NHPs, as well as antisense and ribozyme molecules can also be used in xe2x80x9cgene therapyxe2x80x9d approaches for the modulation of NHP expression. Thus, the invention also encompasses pharmaceutical formulations and methods for treating biological disorders.